Barley glutamyl tRNAGlu reductase: mutations affecting haem inhibition and enzyme activity.
Glutamyl tRNA(Glu) reductase converts glutamate molecules that are ligated at their alpha-carboxyl groups to tRNA(Glu) into glutamate 1-semialdehyde, an intermediate in the synthesis of 5-aminolevulinate, chlorophyll and haem. The mature plant enzymes contain a highly conserved extension of 31-34 amino acids at the N-terminus not present in bacterial enzymes. It is shown that barley glutamyl tRNAGlu reductases with a deletion of the 30 N-terminal amino acids have the same high specific activity as the untruncated enzymes, but are highly resistant to feed-back inhibition by haem. This peptide domain thus interacts directly or indirectly with haem and the toxicity of the 30 amino acid peptide for Escherichia coli experienced in mutant rescue and overexpression experiments can be explained by extensive haem removal from the metabolic pools that cannot be tolerated by the cell. Induced missense mutations identify nine amino acids in the 451 residue long C-terminal part of the barley glutamyl tRNA(Glu) reductase which upon substitution curtail drastically, but do not eliminate entirely the catalytic activity of the enzyme. These amino acids are thus important for the catalytic reaction or tRNA binding.
Vothknecht
U C
UC
Carlsberg Laboratory, Department of Physiology, Copenhagen-Valby, Denmark.
Kannangara
C G
CG
von Wettstein
D
D
D000445:Aldehyde Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001419:Bacteria / Q000201:enzymology / Q000235:genetics; D006418:Heme / Q000494:pharmacology; D001467:Hordeum / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D006418:Heme; D000445:Aldehyde Oxidoreductases; C067282:glutamyl tRNA reductase